The long-term goal of this project is to understand the fundamental aspects of outer membrane vesicles produced by Gram-negative bacteria: How and why are they made? Outer membrane vesicles represent 0.1 to 1% of total outer membrane protein in a growing culture and are produced by all non-pathogenic and pathogenic bacteria studied to date, with virulent bacteria typically producing toxic vesicles and more than non-pathogenic counterparts. Yet the basic processes regarding how and why they are made are unknown. Previously, we have found that outer membrane vesicles contribute a basic stress response of Gram-negative bacteria. We propose that outer membrane vesicle production is governed by specific envelope components and enables remodeling of the periplasmic and outer membrane content in response to environmental changes and stresses. Rapid replacement of cell envelope protein and lipid and vesicle-mediated release of toxic components could be key elements of bacterial survival in the environment, particularly to accommodate stress. Using newly identified vesiculation mutants and biochemistry techniques developed in this laboratory, this research aims to elucidate the mechanism by which vesicles contribute to bacterial adaptation. The research objectives of this study are the following: 1) Characterize the bacterial vesiculation envelope response to stress, 2) Identify and characterize genes in the vesiculation pathway, and 3) Characterize the role of vesicles in cell envelope remodeling. The results will yield long-awaited insight regarding the bacterial processes required for vesicle formation and will reveal details regarding this newly describe stress response. General biological principles of stress product management will be revealed by comparing our findings in the study of bacteria to the related eukaryotic exosomes. The impact on human health is the characterization of a common bacterial process that can be targeted therapeutically to increase bacterial sensitivity to antibiotic treatment. PUBLIC HEALTH RELEVANCE: This project benefits public health by detailing our understanding of bacterial vesiculation, a secretory process common too many pathogenic bacteria that enables bacteria to resist antibiotics and to disperse toxic factors into infected tissues. The proposed research focuses on exposing the molecular basis for vesiculation and aims to facilitate development of this potential therapeutic target.